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BE 167L Lecture 5 General

BE 167L Lecture 5 General - BE 167L – Bioengineering...

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Unformatted text preview: BE 167L – Bioengineering Lab Lecture 5 Today •  Quiz 1 overview •  Why Do Cell Culture? •  Mammalian Cell Culture •  Cell Assay Techniques Upcoming Thursday: Three ­dimensional Cell Culture and the Cellular Microenvironment Next Tuesday: Case Study 3 Quiz 1 stats •  70 points total •  Low – 16 (23%) •  High – 66 (94%) •  Mean – 45.6 (65%) •  Median – 45 (64%) •  SD – 11 Problem areas •  Kohler Illumina[on •  Amino acid reac[vity •  Importance of fluorescence spectrum overlap •  Designing scien[fic experiments •  Misconcep[ons about the use of stamps with cells What is Kohler illumina[on and how do you adjust it? •  A way to create uniform ligh[ng on the sample •  If ligh[ng is not uniform this should be one of the first things you check •  Some microscopes allow you to focus the condenser by moving it up and down un[l the light is defocused. The aperture should also be centered. •  Some microscopes have automa[c kohler illumina[on fixed, so if it is off, you may have to call to have it fixed. •  Always try to read the manual of any microscope you will use frequently to learn how to troubleshoot these issues •  Focusing the condenser in brighdield •  Aligning the phase rings for phase contrast Carboxylic acids aren’t good nucleophiles •  You have to put a leaving group on it to ac[vate it •  Good nucleuophiles will displace them •  COO ­ doesn’t displace NHS •  All ionizable amino acids don’t undergo the same reac[ons Fluorescence is a con[nuous spectrum, make sure there are no cri[cal overlaps •  Spectral overlap of the abs/ems can create unintended signals •  Depending on your filters and camera you may see more wavelengths than you think •  Emission from one and absorp[on by another can contribute incorrect signals How do you design a complete scien[fic experiment? (mini ­projects this quarter) •  Define a hypothesis to be tested •  Outline experimental condi[ons that test it •  Choose appropriate control condi[ons •  Decide on an acceptable way to replicate data for consistency •  Define methods of collec[ng data and analyzing that data to address the hypothesis •  An[cipate different outcomes and the meaning of possible data analysis How and when should you use a PDMS stamp? •  Stamping requires drying the par[cle onto the PDMS ­ which would kill cells •  Stamping requires applying a weight and leaving it (dry) for several minutes – which would kill cells underneath it •  Stamping achieves a pajern, if no pajern is really necessary you can achieve protein adsorp[on by just soaking in a solu[on, like how it got onto the PDMS •  There should be an experimental reason for the pajern being stamped (increase efficiency or study interac[ons…) Reasons for Cell Culture •  Produce therapeu[c protein products – Example: Epogen •  Study of cells and cell systems in a controlled manner – Signaling, Trafficking, Migra[on, Apoptosis •  Determine effects of novel drugs (e.g. cytotoxicity, metabolism, efficacy of an[ ­ cancer drugs) EPOGEN® (from Amgen) •  Recombinant human erythropoie[n – Replenishes red blood cells •  Grown in roller bojles (CHO cells) – Two ­dimensional cell culture – Have a warm room with a lot of roller bojles 12 Growing Cells Outside the Body 3. Determine correct culture conditions 4. Scale-up 1. Obtain tissue 2. Obtain pure cell population Primary vs. Immortalized Cells •  Primary Cells – Grow, but lose func[on or die aoer a limited number of cell divisions or [me •  Immortalized Cells – Can grow indefinitely, may accumulate gene[c changes over [me •  May have cancerous origin •  Modified primary cell to become immortalized HeLa Cells (Henrieja Lacks) •  You will be working with the HeLa mammalian cell line. – Purchased from American Type Culture Collec[on (ATCC), Catalog # CCL ­2. – This cell line is a virus ­transformed human epithelial cell line from a cervical carcinoma. 15 How can we grow cells in vitro? •  What nutrients are necessary? – Medium, fetal bovine serum – Different for different cell lines •  What physical condi[ons are necessary? – Temperature (37 C) – Controlled humidity •  How do we get rid of waste? – Media exchange, large media volumes, passaging •  Cells can change over [me (culture adapta2on) Medium for Growing HeLa Cells •  “Blank Medium” –  Minimum Essen[al Medium (MEM) •  Invitrogen, SKU# 41500 ­034 •  Contains Earle's salts, L ­glutamine, nonessen[al amino acids, and phenol red, but no sodium bicarbonate. –  Sodium bicarbonate –  Sterile filter with a 0.22 µm filter. •  “Complete Medium” –  “Blank Medium” –  Fetal bovine serum (FBS) –  Penicillin and streptomycin –  Sodium pyruvate 17 Fetal Bovine Serum •  Contains several components. –  If your experiment is performed in the presence of serum and may be strongly dependent on its components, you probably want to use the same lot. –  If your experiment is performed serum ­free or you know its dependence on serum is not as strong, you should at least purchase lots of FBS that are similar. •  You can get some free samples to test out the cell morphology. •  Due to concerns with mad cow disease, biotechnology companies are trying to establish serum ­free medium formula[ons. 18 Sodium Bicarbonate •  The mammalian cells are grown in an incubator with moisture (usually generated by a pan of autoclaved water) and 5% CO2. •  Use sodium bicarbonate (NaHCO3) as the buffer. – NaHCO3 Na+ + HCO3 ­ – HCO3 ­ + H2O H2CO3 + OH ­ – H2CO3 H2O + CO2 (gas) •  If there is 5% CO2 in the air, it will keep NaHCO3 in solu[on. 19 Phenol Red •  Phenol red, or phenolsulfonphthalein, is used as a pH indicator. •  As the media goes acidic, it will go from red to orange to yellow. •  As the media goes basic, it will go from red to dark red to purple. •  If you leave the cap of your media open for a long [me in the biosafety cabinet (which you shouldn’t do anyway for sterility reasons), what color will your media turn? 20 Buffers for Atmosphere •  Several different buffers exist for performing experiments exposed to the atmosphere. – Phosphate buffers (monobasic and dibasic) – HEPES – Tris – Etc. •  Can add these different buffer salts to MEM (instead of sodium bicarbonate) if performing experiments for a short period of [me not in the incubator. 21 Passaging •  Do not want the cells to become too crowded. –  Will get contact inhibi[on (idea of confluency). •  For some cell lines (not HeLa cells), the split ra[o cannot get lower than 1:3 or 1:4. –  Need cell ­cell interac[ons. •  Trypsin –  Will cut the proteins that bind the cells to the flask. –  Predominantly cleaves pep[de bonds at carboxy side of Arginine and Lysine. –  There is trypsin inhibitor in FBS. •  EDTA –  Will chelate divalent metals that enhance the binding of proteins to surfaces and each other (clumping). 22 Hemocytometer 23 A B C 1 mm D http://fig.cox.miami.edu/~ddiresta/BIL151/CellCounts.ppt Hemocytometer •  There are 9 large squares, including A through D. – Each square is 1 mm x 1 mm. – Only count in four of the squares (i.e. A through D). •  Depth of the coun[ng chamber is 0.1 mm. – Volume of each large square is therefore 1 mm2 x 0.1 mm = 0.1 mm3. •  Unit conversion to be used later 24 – 1 ml = 1 cm3 = (10 mm)3 = 1000 mm3 Live Dead Stain •  Live (Green) – Calcein AM enters cell membrane – Cleaved by esterases in living cells – Resultant product fluoresces green •  Dead (Red) – Ethidium homodimer cannot enter intact membranes – i.e. cannot enter live cells – Cell membrane becomes leaky when cell dies 25 – Ethidium homodimer binds DNA and fluoresces red Propidium iodine Calcein AM Ethidium homodimer 26 Time for Calcein AM Staining ...
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